Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.

The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.RNA degradation ͉ RNA processing ͉ x-ray crystallography ͉ RNase D T he RNA exosome participates in a wide range of reactions, including processing and degradation of tRNA and rRNA as well as degradation of both nuclear and cytoplasmic RNA polymerase II-derived transcripts (1-6). The core eukaryotic exosome is present in both the cytoplasm and nucleus and consists of 10 proteins, with at least 7 harboring proven or predicted 3Ј-to-5Ј exonuclease activity (one RNase II and six RNase PH type) (7-10). In the yeast nucleus, the complex is distinguished by three additional proteins, the RNase D-type enzyme Rrp6p (for ribosomal RNA processing), the DEAD-box RNA helicase Mtr4p, and the less well characterized protein Rrp47p (7, 11). Exosomes are found in both eukaryotes and archaea, and, recently, several crystal structures of archaeal subcomplexes were reported (12)(13)(14). The center of the archaeal exosome consists of three Rrp41 and three Rrp42 proteins forming an overall donut-shaped heterohexameric structure. Rrp41 and -42 are each similar to three proteins in the eukaryotic complex, which consists of six different proteins forming the ''donut' ' (12, 14). This ring-like structure is able to bind additional proteins, forming a ''cap'' suggested to constrict and probably regulate the entry of RNA into the central cavity containing the phosphorolytic active sites (12).The nuclear exosome is essential for maturation of eukaryotic ribosomal RNAs (25S, 18S, and 5.8S), which are synthesized as a single transcript (for a review, see ref. 15). Processing is initiated by endonucleolytic cleavage of the external transcribed spacers (ETSs), which are subsequently degraded by Rrp6p (16). This protein is also required for trimming of the two internal transcribed spacers 1 and 2 during maturation to produce the mature rRNAs, and a 30-nt 3Ј-end extended form of 5.8S rRNA appears in ⌬rrp6 cells (17). Similarly, many small nucleolar RNAs (snoRNAs) depend on the exosome during their maturation (18,19), and deletion of Rrp6p in yeast also leads to accumulation of extended forms of both polycistronic snoRNAs (18) and the independently transcribed snoRNAs, such as snR33 and snR40 (20).Rrp6p is homol...

Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but so far not in archaeal viruses, despite the near ubiquity of CRISPR-Cas systems in archaea. Here, we report the functional and structural characterization of two archaeal Acrs from the lytic rudiviruses, SIRV2 and SIRV3. We show that a 4 kb deletion in the SIRV2 genome dramatically reduces infectivity in Sulfolobus islandicus LAL14/1 that carries functional CRISPR-Cas subtypes I-A, I-D and III-B. Subsequent insertion of a single gene from SIRV3, gp02 (AcrID1), which is conserved in the deleted fragment, successfully restored infectivity. We demonstrate that AcrID1 protein inhibits the CRISPR-Cas subtype I-D system by interacting directly with Cas10d protein, which is required for the interference stage. Sequence and structural analysis of AcrID1 show that it belongs to a conserved family of compact, dimeric αβ-sandwich proteins characterized by extreme pH and temperature stability and a tendency to form protein fibres. We identify about 50 homologues of AcrID1 in four archaeal viral families demonstrating the broad distribution of this group of anti-CRISPR proteins.

Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3′–5′ exonucleases potentially responsible for the deadenylation reaction. Here, we present the crystal structure of the Pop2p subunit from Schizosaccharomyces pombe determined to 1.4 Å resolution and show that the enzyme is a competent ribonuclease with a tunable specificity towards poly-A. In contrast to S. cerevisiae Pop2p, the S. pombe enzyme contains a fully conserved DEDDh active site, and the high resolution allows for a detailed analysis of its configuration, including divalent metal ion binding. Functional data further indicates that the identity of the ions in the active site can modulate both activity and specificity of the enzyme, and finally structural superposition of single nucleotides and poly-A oligonucleotides provide insight into the catalytic cycle of the protein.

SummaryPhosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected selfhomologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown.Phosphonate compounds that contain a stable carbon-phosphorus (C-P) bond are utilised as a source of phosphate by microorganisms in many natural environments where the low levels of free and organic phosphate limit growth 1 . The C-P lyase pathway, which converts phosphonate into 5-phosphoribosyl-α-1-diphosphate (PRPP) in an ATP-dependent fashion, is activated upon phosphate starvation in many bacterial species including Escherichia coli 2,3 . The enzymes of this pathway have a very broad substrate specificity enabling the Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http:// www.nature.com/authors/editorial_policies/license.html#terms 3 Correspondence and requests for materials should be addressed to D.E.B.(deb@mbg.au.dk, phone +45 21669001). Author Contributions. P.S., L.A.P., B.H.J., B.J., and D.E.B. designed and P.S., L.B.V., C.J.R, and B.J. carried out the experiments. P.S., M.K., and D.E.B. determined the crystal and EM structures while C.J.R. and L.A.P. carried out final refinement of the EM structure as well as EM structure validation. P.S, M.K., C.J.R., L.A.P., B.H.J., B.J., and D.E.B. wrote the manuscript.Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession code 4XB6. The EM density map has been deposited in the Electron Microscopy Data Bank (EMDB) with accession code EMD-3033.